Cancer cells typically display abnormal genomes with aneuploidy and chromosomal rearrangements, including high frequency gene amplification at late stages of tumor progression. The existence of a number of human cancer prone genetic diseases with defects in DNA repair, whose non-tumorous cells display unstable karyotypes provides strong evidence that genomic instability is heritable, and associated with a predisposition to cancer.
The development of a malignant cell is a multistep process. The spontaneous rate of mutation in normal somatic cells is less than 10.sup.-5 mutation/gene/generation. Thus, the accumulation of a number of genetic changes by a clone of cells would be more likely to occur if an early step produces a genetically unstable cell. Eukaryotes have generated a variety of mechanisms for limiting the formation of abnormal, heritable genetic changes. These include mechanisms for maintaining fidelity of DNA replication and segregation, mechanisms for DNA repair, and checkpoint genes for cell cycle progression or for programmed cell death (apoptosis).
It has been assumed that one mechanism leading to genomic instability could be a defect in a DNA repair pathway. The recent findings of a defective mismatch repair in hereditary non-polyposis colorectal cancer confirmed that assumption.
Another possible mechanism for generating genomic instability is perturbation of the normal cell cycle control. At least two stages in the cell cycle are regulated in response to DNA damage--the G.sub.1 -S and the G.sub.2 -M phase transitions. These transition serve as checkpoints at which cells delay cell cycle progression, presumably to allow repair of damage before the cell enters either replicative DNA synthesis (G.sub.1 -S), when damage could be perpetuated, or before the cell enters mitosis (G.sub.2 -M), when chromosomal breaks would result in the loss of genetic material.
This possibility was initially supported by features of Barrett's esophagus, a cancer-prone syndrome, which shows a sequential appearance of high S and/or high G.sub.2 phase populations, followed by the emergence of aneuploid cells and subsequently the development of tumors. The demonstration by Weinert and Hartwell that mutations in the yeast radiation-monitoring G.sub.2 -M checkpoint gene RAD9 (Hartwell and Weinert, 1989) and in the G.sub.1 -S checkpoint genes RAD5 and RAD51 lead to increased spontaneous chromosome loss further supported this supposition. These observations led Hartwell to propose that some of the tumor suppressor genes actually operate as cell cycle checkpoint genes. The prediction was soon proven correct when Kastan and colleagues demonstrated that human p53 is a G.sub.1 -S checkpoint gene which prevents entry into S phase when DNA is damaged by irradiation (Kastan et al., 1991; Kuerbitz et al., 1992). This result becomes more noteworthy when we take into account that the loss or mutation of the p53 gene is the most common alteration found in sporadic, non familial cancers of either solid tumor or hematopoietic ones. Furthermore, p53 also protects the cell from genomic instability reflected in gene amplification and acts as a checkpoint by activating an inhibitor (p21) of G.sub.1 -S phase transition (El-Deiry et. al., 1993; Dulic et. al., 1994).
Another example of the involvement of aberrations in cell cycle control with human cancers is the recognition that an inhibitor (p16) of cyclin-dependent kinase 4 is encoded by a tumor suppressor gene, which is defective in various human cancers including melanoma.
A further demonstration of the link between human cancers and the cell cycle machinery has been the finding that an oncogene involved in parathyroid adenoma (PRAD1) and B-cell lymphoma (bcl-1) actually encodes cyclin D1. Moreover, the cellular proteins sequestered by pRb include, in addition to the E2F transcription factor, cyclin D1 and perhaps cyclin D3. Phosphorylation of pRb by cdk2/cyclin E releases the cyclins D1/D3 from the complex, allowing them to associate with cdk(s) and phosphorylate "S-phase targets".
More recently it was shown that the fission yeast DNA damage monitoring G2 checkpoint genes rad24 and rad25 encode 14-3-3-protein homologs which associate with the oncogenic middle tumor antigen of murine polyoma virus.
Thus, the relationship between human cell-cycle checkpoint genes and human cancers has been firmly established. The role of p53 in DNA damaged-induced G1 arrest has resulted in this gene being considered as "the guardian of the genome". Seemingly this role of p53 in preventing mutations and chromosomal rearrangements or loss could have been sufficient to explain its tumor suppressor activity and its deletion/disregulation in over half of human cancers. However, over the past few years, another important function of p53 has been discovered and this is its requirement for apoptosis induced by radiation (UV as well as .gamma.-rays), anti-neoplastic DNA damaging agents, oncogenes activation and withdrawal of hematopoietic survival factors (Yonish-Rouach et. al., 1991; Clarke et. al., 1993; Lowe et. al., 1993).
Thus p53 acts as a tumor suppressor gene by either causing a cell cycle arrest or by the induction of apoptosis. The mode of action of p53 at this "decision fork" depends on multiple factors which constitute the cellular milieu. Notable effectors are the tumor suppressor gene Rb which acts as a G.sub.1 cell cycle checkpoint gene (repressor for S phase entry) as well as an anti-apoptotic gene whose disregulation is countered by p53 mediated apoptosis. Likewise it appears that the adenovirus E1A and c-myc onco-proteins sensitize cells deprived of growth factors to undergo apoptosis through sequestering of the pRb anti-apoptotic protein.
Another effector which counters apoptosis is the Bcl-2 proto-oncogene. Bcl-2 like p53 responds to growth factor withdrawal, DNA damage and oncogene activated apoptosis.
The classical dogma that antineoplastic agents cure cancer by selectively killing rapidly diving cells has proved to be incorrect. As outlined above, some oncogenes which induce entry into the cell cycle do in fact predispose cells undergoing untimely proliferation (for example under conditions of limited growth factor supply) to programmed cell death. Thus, due to varying apoptosis thresholds, radiation/chemotherapy may induce apoptosis in the tumor cell and merely a cell cycle arrest in the surrounding normal cells. Accordingly the success of tumor eradication by anti-neoplastic agents is critically dependent on the cellular balance between apoptotic and anti-apoptotic processes. One of the promising approaches to lower the threshold for radiation/chemotherapy-induced apoptosis would be to ablate the activity of anti-apoptotic gene(s) in the tumor, thereby curing the malignancy.
Few examples of natural antisense RNAs have been published. It is noteworthy, however, that natural antisense RNAs must have been conserved during evolution, and that the few mammalian examples known so far involve genes such as p53 and myc whose expression is essential for growth control.